DNA sequences involved in the heavy chain switch

描述（由申请人提供）：抗体生产中的重链开关对于有效保护病原体至关重要。重链开关是由开关区域之间的DNA重组事件介导的。开关区是位于每个重链基因上游的2-8 kb的简单序列串联重复延伸[三角洲除外]。两个开关区之间的重组必须是有效的，这样才能产生大量的特定类型的抗体。开关重组的效率被认为是由位于重链常数区位点3'的四个增强元件调控的。在目标1和目标2中，我们将测试四种增强子元件的不同组合的删除如何改变开关重组的效率。七种类型的分泌抗体中的每一种都是为最佳中和，清除或研磨病原体子集而设计的。因此，重链开关必须以一种重链特异性的方式进行调节，以便产生对给定病原体具有最佳保护作用的重链。在重链开关发生之前，特定重链基因的染色质打开，这可以通过开关重组事件前种系转录本的表达来最好地测量。种系转录和开关区之间的DNA重组受细胞因子调控。在Aim 3中，我们将确定细胞因子对生殖系转录和特定基因的开关重组的调节是由生殖系转录物启动子区域的DNA序列还是由开关区域序列调节的。所有三个目标的实验将通过测试整个重链恒定区基因座的转基因的各种突变形式来进行。与其他方法相比，在大肠杆菌中突变重组DNA分子，然后测试其介导种系转录和在正常B淋巴细胞中切换所有七个转基因重链基因重组的能力，具有几个优势。调节重链转换是几个实际免疫学问题的中心。疫苗的设计应能引起最大量的最有利类型的抗体。几乎所有的过敏都是由一种抗体的表达引起的。从提出的实验中获得的理解可能会改进疫苗开发和过敏治疗的方法。

DESCRIPTION (provided by applicant): The heavy chain switch in antibody production is critical to effective protection against pathogens. The heavy chain switch is mediated by a DNA recombination event between switch regions. Switch regions are 2-8 kb long stretches of simple sequences repeated in tandem that lie upstream of each heavy chain gene [except delta). Recombination between two switch regions must be efficient so that large quantities of a given type of antibody can be produced. The efficiency of switch recombination is thought to be regulated by four enhancer elements that lie 3' of the heavy chain constant region locus. In Aims 1 and 2, we will test how deletion of various combinations of the four enhancer elements alter the efficiency of switch recombination. Each of the seven types of secreted antibodies are designed for the optimal neutralizing, clearing, or milling of a subset of pathogens. Hence, the heavy chain switch must be regulated in a heavy chain-specific manner in order to produce a heavy chain for the best protection against a given pathogen. The heavy chain switch is preceded by opening of the chromatin for a specific heavy chain gene, which is best measured by the expression of germline transcripts before the switch recombination event. Germline transcription and DNA recombination between switch regions are regulated by cytokines. In Aim 3, we will determine if cytokine regulation of germline transcription and switch recombination to a specific gene is regulated by DNA sequences in the promoter region for germline transcripts or by the switch region sequences. The experiments in all three Aims will be carried out by testing various mutant forms of a transgene of the entire heavy chain constant region locus. The ability to mutate this recombinant DNA molecule in E. coli, and then test its ability to mediate germline transcription and switch recombination of all seven transgenic heavy chain genes in normal B lymphocytes, offers several advantages compared to other approaches. Regulated heavy chain switching is at the center of several practical immunological problems. Vaccines should be designed to elicit the largest amounts of the most favorable types of antibodies. Almost alll allergies are caused by the expression of one type of antibody. The understanding to be gained from the proposed experiments may improve approaches toward vaccine development and toward treatment of allergy.